Overexpression of programmed death ligand 1 enhances immunosuppressive capacity of human umbilical cord mesenchymal stromal cells against T cells

Abstract

BACKGROUND: The T cell immunosuppressive activity of mesenchymal stromal cells offers new hope for the treatment of autoimmune diseases. Amimatoside injection, a drug of human umbilical cord mesenchymal stromal cells, has been approved for the treatment of acute graft-versus-host disease (GVHD) primarily affecting the digestive tract, after steroid therapy failure, in patients aged 14 years and older. Therefore, further exploration of the T cell immunosuppressive potential of mesenchymal stromal cells can lay a foundation for the treatment of autoimmune diseases. OBJECTIVE: To investigate the effect of programmed death ligand 1 (PD-L1) gene overexpression on the inhibition of CD4+ T cell proliferation by human umbilical cord mesenchymal stromal cells (hUC-MSCs). METHODS: (1) hUC-MSCs were cultured in vitro to passages 0, 1, 2, and 3, and the percentage of PD-L1-positive cells was detected by flow cytometry. (2) hUC-MSCs were divided into experimental group and negative control group. The experimental group was transduced with lentivirus-mediated PD-L1 gene, while the negative control group was transduced with lentivirus carrying empty plasmid vector. Transfection efficiency was assessed by flow cytometry, real-time quantitative PCR, and western blot. (3) CD4+ T cells were enriched from healthy human peripheral blood using magnetic beads, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and co-cultured with hUC-MSCs from the experimental and negative control groups at a ratio of 5:1. The proportion of CFSE-dim CD4+ T cells was detected by flow cytometry. (4) RNA sequencing was performed on hUC-MSCs from both groups, and single-cell RNA sequencing was performed on the experimental group. Subgroups were identified based on function, and bioinformatics analysis was used to depict heatmaps of marker genes, enriched signaling pathways, and gene regulatory networks for each subgroup. RESULTS AND CONCLUSION: (1) The percentage of PD-L1-positive cells in hUC-MSCs gradually decreased with increasing passage number. (2) hUC-MSCs stably overexpressing PD-L1 were successfully constructed, with significantly increased PD-L1 expression in the experimental group. (3) Transcriptome sequencing data suggested that PD-L1-overexpressing hUC-MSCs promoted the upregulation of genes related to immune effector regulatory pathways. (4) Co-culture of CD4+ T cells with hUC-MSCs showed a significant decrease in the proportion of CD4+ T cells in the experimental group. (5) Based on single-cell RNA sequencing results, PD-L1-overexpressing hUC-MSCs could be divided into three functional subgroups with heterogeneity; PD-L1 gene expression was higher in subgroup 1, and the significantly high expression of histone methyltransferase SETDB1 might be closely related to enhanced T cell immunosuppressive function. These findings indicate that PD-L1 gene overexpression can significantly enhance the T cell immunosuppressive capacity of hUC-MSCs. Single-cell RNA sequencing can effectively classify PD-L1 gene-modified hUC-MSCs based on their functional characteristics, providing theoretical support for improving the clinical efficacy of hUC-MSCs.

1. Introduction

Autoimmune diseases are pathological conditions caused by the immune system mounting an immune response against self-components. The normal outcome of an immune response to foreign antigens is antigen clearance, but when the immune system responds to self-cell or tissue antigens, the self-cells or tissues are not completely eliminated by effector cells but are continuously attacked, leading to a disease state with a rising incidence [1-4]. Current treatments, such as glucocorticoids, immunosuppressants, and biologics, often face challenges including insufficient efficacy, high relapse rates, and significant long-term toxicity, failing to meet clinical needs [5-8].

Mesenchymal stromal cells (MSCs) offer a new strategy for treating autoimmune diseases due to their potent immunomodulatory and tissue repair capabilities [9-11]. Human umbilical cord-derived MSCs (hUC-MSCs) are particularly promising because of their abundant source, lack of ethical controversy, strong proliferative capacity, and low immunogenicity [12-14]. MSC therapy has achieved initial clinical success; for example, Amimatoside injection (umbilical cord-derived) has been approved in China for treating specific types of acute graft-versus-host disease [15]. However, the functional heterogeneity and instability of key immunomodulatory molecule expression during in vitro expansion of hUC-MSCs remain significant limitations.